Metal recovery and metal remediation and the associated need for efficient and safe methods for clean up of metal waste is a continuing environmental and business concern due to the toxicity and potential risk to human health posed by metal contaminants, as well as the economic value of precious heavy metals. Indeed, as the discharge of toxic wastes from agricultural, industrial and other commercial operations continues, the need for effective, safe and low-cost metal remediation methods increases. In a recent report by the United States Environmental Protection Agency (US EPA), metal contamination remains and historically has been a key concern at many contaminated sites (US EPA Work Assignment #011059, Mar. 5, 1997, Contract #68-W5-0055). In addition, there are numerous published reports of damage to wildlife, livestock, plant life as well as danger to human health as a result of metal poisoning from contaminated soil or waste matter (Impact of Lead-Contaminated Soil on Public Health by Xintaras, C. May 1992 at http://www.atsdr.cdc.gov/cxlead.html). For example, a primary concern to humans is the health hazard created by lead (Pb) contamination. Exposure to lead can occur through a variety of methods such as by ingestion of lead from food, water, soil, or even inhalation of dust. Lead poisoning is extremely dangerous and potentially fatal, with symptoms including seizures, mental retardation and behavioral disorders. Therefore, methods for metal remediation are extremely valuable both for their protection of our environment as well as for protection from diseases.
Recovered metals from various waste, discard or recycling efforts provide immense economic value as well as augmenting environmental pollution control. Metal recovery can be from innumerable and varied sources such as from waste electronic devices (transistors, chips, transformers, bus bars, cathodes, and microprocessors, populated computer circuit boards PCBs, motherboards). Costs associated with hazardous disposal of industrial waste in the absence of metal reclamation are enormous. Therefore, metal recycling or reuse of metal extracted from scrap or discarded metal-containing items not only reduces the volume and cost of metal waste requiring specialized disposal and handling efforts, but the reclaimed metal can also be resold or reused to provide additional economic value.
Prior art attempts at treating metal contamination have traditionally employed cleanup technologies which consist primarily of physically removing and then disposing of contaminated matter. These methodologies are not only labor intensive and less efficient, but also carry a high expense associated with removal and disposal of large or bulk quantities of contaminated waste. Metal contamination is especially difficult to remediate because unlike other types of waste such as chemical or organic matter, metals cannot be directly destroyed or converted. For example, current technologies for remediating metal contaminated soils consist primarily of landfilling or soil excavation with physical or chemical separation of the metal contaminants. Treatment of contaminated ground water usually involves flushing, filtration or chemical extraction to remove the contaminating metals. As a result, the cost of soil or ground water remediation is high, ranging in the hundreds to thousands of millions of dollars in projected five-year costs per site (U.S. EPA, 1993).
In addition, the risk to humans and the environment from heavy metal contamination is not limited to soil or ground water, but also includes other sources such as industrial waste, sludge waste, wastewater, radioactive waste (such as radionuclides from research and medical waste) and mining waste. Depending on the physical and chemical form of the metal contaminant to be removed, as well as the cost-benefit analysis for a particular remediation approach, which of the existing technologies is better suited for a particular site will vary. However, due to the high cost of traditional cleanup technologies, there still remains a great need for a less-expensive, safe and effective heavy metal recovery and cleanup technology.
There are some technologies currently available for the recovery or remediation of heavy metal contaminated waste. In general, these technologies combine one or more of the following general approaches: isolation, immobilization, toxicity reduction, physical separation or extraction of metal contamination from a waste product. Isolation technologies utilize a containment strategy in an attempt to confine a contaminated site or area so as to prevent further spread of the toxic metal waste. Immobilization technologies reduce the mobility of metal contaminants and include systems which provide an impermeable barrier to separate underlying layers of soil (containing the metal contaminants) from the topsoil layer. Also used are physical barriers which restrict the flow of uncontaminated groundwater through a contaminated site. Additionally, there are toxicity reduction processes which generally use chemical or biological techniques to decrease the toxicity or mobility of metal contaminants. Included in toxicity reduction processes are biological treatment technologies, which apply newer biotechnical approaches.
Metal remediation is a relatively new application of biological treatment technologies and includes processes such as bioaccumulation, phytoremediation, phyotextraction, and rhizofiltration. All of these biological treatments use certain plants and microorganisms to remediate metals through either adsorption, absorption, or concentration of contaminating metal ions. For example, in bioaccumulation, plants or microorganisms actively take up and accumulate metals from contaminated surroundings.
In phytoremediation, specific plants that have developed the ability to selectively remove metal ions from soil are used. Such plants include certain “hyperaccumulator” species such as the alpine pennycrass plant, which is capable of accumulating metals at levels of 260 times greater than most plants before showing toxicity symptoms. Most hyperaccumulator plants, however, are very slow growing and have specific growth requirements. Some of these growth requirements are not conducive to the use of these plants at sites or in situations where metal recovery or remediation is needed. Furthermore, there are very few plant species known or available for recovery or remediation use. Therefore, given the persistent and high incidence of metal contamination at environmental and waste sites (about 75% of Superfund Sites contain metal ions as a form of contamination, U.S. EPA, 1996), more efficient methods and approaches for removing heavy metals from contaminated sources are still needed.
More recently, in an attempt to meet these needs, biotechnological approaches have been employed as an alternative strategy to metal recovery and remediation. Included in these biotechnology approaches are the use of tobacco plants that have been manipulated to express metallothionein genes (Maiti et al. Seed-transmissible expression of mammalian metallothionein in transgenic tobacco, Biochem Biophys Res Commun. 150(2):640-7,1988). Metallothioneins (MTs) are small metal binding proteins ubiquitously distributed throughout the animal kingdom. They have high metal binding affinities and are believed to be important in controlling the intracellular levels of free metal ions. However, little else is known about their function or biological purpose. Metallothioneins were first discovered in 1957 in horse tissue. Since then, they have been identified in species ranging from fungi and shellfish to mice and humans.
The structural features of MTs include a high cysteine composition and lack of aromatic amino acids. The cysteine residues are responsible for the protein's high affinity metal ion binding capabilities. In general, MTs have a high degree of amino acid sequence similarity. However, the proteins or known gene sequences encoding the proteins have been used primarily in either the research setting or in disease treatment methodologies.
Accordingly, one of the objects of the present invention is to provide novel metal binding proteins for the removal of metals from a variety of substrates. This technology would allow for the efficient, cost effective, safe and simple removal of heavy metals from environmental waste or other materials contaminated with heavy metal.